Actin is among the three highly conserved, highly abundant and highly important protein families in eukaryotic cells together with histones and tubulins. Actin can account for as much as 10% of total cellular proteins and is central in numerous functions in all eukaryotes involving motility, such as organelle movement, cell division, cell migration, cell shape maintenance, muscle contraction, nerve growth and many other functions critical to human health. However, the functions of actin in the cell nucleus have remained controversial for decades due to the lack of tractable systems to study nuclear actin. We have built a defined genetic and biochemical system in yeast to study the mysterious nuclear actin. This system allows us to define a distinct monomeric actin mechanism in the INO80 chromatin remodeling complex, which contrasts the established mechanism of cytoplasmic actin based on polymerization. Through continued study of the actin subunit in the INO80 complex, we have made a breakthrough discovery that actin undergoes a system of post-translational modification (PTMs) similar to histones. Given the numerous functions of actin in all eukaryotes, actin must be regulated to target distinct biological processes. The established paradigm that explains actin's participation in such a diverse range of functions is that actin can be directed to specific functions through interactions with actin- binding proteins (ABPs). Indeed, hundreds of ABPs involved in regulating actin biology have been identified. Our discovery of actin PTMs has led us to propose a new paradigm of actin regulation through actin PTMs. We hypothesize that distinct actin PTMs provide a major new mechanism for regulation of actin functions in biological processes, such as in transcription. Our initial study on actin R256 mono-methylation (R256me1) provides a validation of the actin PTM hypothesis, as R256me1 represents the first evolutionarily conserved mark for nuclear actin. Furthermore, actin R256me1 is implicated in transcription. Strikingly, mutations in the actin R256 residue (R258 in human) have been shown to cause human diseases, such as Thoracic Aortic Aneurysm and Dissection (TAAD), raising the possibility that actin PTMs contribute to TAAD through transcription. In the long term, we would like to establish the actin PTM hypothesis and discover its links to human diseases. In this proposal, we focus on the study of actin R256me1 in order to build the foundation for the actin PTM hypothesis. We propose to use genetic and biochemical approaches to define the enzymology of actin R256me1. Furthermore, we will investigate the biological roles of actin R256me1 in transcription and chromatin modification. Importantly, we will also determine the contribution of nuclear actin and actin PTMs to TAAD. The implications of the proposed studies are far reaching, as these pioneering actin PTM studies will open a new field in basic research and also provide a novel direction in medical research. Similar to the studies of histone PTMs, which exploded in the 1990s and revolutionized chromatin biology, we believe that the studies of nuclear actin and actin PTMs will usher in a new era in chromatin and actin biology.